The role and mechanism of tumor metastasis-associated gene 1 in radiosensitivity of HeLa cells / 中华肿瘤杂志

Objective: To determine the effect of tumor metastasis-associated gene 1 (MTA1) on the sensitivity of HeLa cells to radiotherapy, and to clarify its molecular mechanism. Methods: The transcriptome differences between MTA1 knocked down Hela cells and control cells were analyzed, and the differentially expressed genes (DEGs) was used to perform Gene-Set Enrichment Analysis (GSEA) and Gene Ontology (GO) cluster analysis. Flow cytometry was used to detect apoptosis in MTA1-overexpressed HeLa cells and control cells before and after 10 Gy X-ray irradiation. Cloning formation assay and real-time cellular analysis (RTCA) were used to monitor the cell proliferation before and after 2 Gy X-ray irradiation. To dissect the underlying molecular mechanisms of MTA1 affecting the sensitivity of radiotherapy, the proteins encoded by the DEGs were selected to construct a protein-protein interaction network, the expression of γ-H2AX was detected by immunofluorescence assay, and the expression levels of γ-H2AX, β-CHK2, PARP and cleaved caspase 3 were measured by western blot. Results: By transcriptome sequencing analysis, we obtained 649 DEGs, of which 402 genes were up-regulated in MTA1 knockdown HeLa cells and 247 genes were down-regulated. GSEA results showed that DEGs associated with MTA1 were significantly enriched in cellular responses to DNA damage repair processes. The results of flow cytometry showed that the apoptosis rate of MTA1 over-expression group (15.67±0.81)% after 10 Gy X-ray irradiation was significantly lower than that of the control group [(40.27±2.73)%, P<0.001]. After 2 Gy X-ray irradiation, the proliferation capacity of HeLa cells overexpressing MTA1 was higher than that of control cells (P=0.024). The numbers of colon in MTA1 over-expression group before and after 2 Gy X-ray irradiation were (176±7) and (137±7) respectively, higher than (134±4) and (75±4) in control HeLa cells (P<0.05). The results of immunofluorescence assay showed that there was no significant expression of γ-H2AX in MTA1 overexpressed and control HeLa cells without X-ray irradiation. Western blot results showed that the expression level of β-CHK2 in MTA1-overexpressing HeLa cells (1.04±0.06) was higher than that in control HeLa cells (0.58±0.25, P=0.036) after 10 Gy X-ray irradiation. The expression levels of γ-H2AX, PARP, and cleaved caspase 3 were 0.52±0.13, 0.52±0.22, and 0.63±0.18, respectively, in HeLa cells overexpressing MTA1, which were lower than 0.87±0.06, 0.78±0.12 and 0.90±0.12 in control cells (P>0.05). Conclusions: This study showed that MTA1 is significantly associated with radiosensitivity in cervical cancer HeLa cells. MTA1 over-expression obviously reduces the sensitivity of cervical cancer cells to X-ray irradiation. Mechanism studies initially indicate that MTA1 reduces the radiosensitivity of cervical cancer cells by inhibiting cleaved caspase 3 to suppress apoptosis and increasing β-CHK2 to promote DNA repair.

Juglone induces proliferation inhibition and apoptosis of cervical cancer cells via promoting c-Myc ubiquitination / 南方医科大学学报

OBJECTIVE@#To observe the expression of c-Myc protein in cervical cancer HeLa cells and explore the effect of juglone on the proliferation and apoptosis of HeLa cells by affecting c-Myc ubiquitination.@*METHODS@#HeLa cells treated with different concentrations (0, 10, 20, or 50 μmol/L) of juglone or with 20 μmol/L juglone for different time lengths were examined for expression of c-Myc protein with Western blotting. The half-life of c-Myc protein was determined using cycloheximide (CHX) and c-Myc protein degradation was detected using coimmunoprecipitation. We also assessed the effects of 20 μmol/L juglone combined with 0, 1.0 or 2.0 μmol/L MG132 (a proteasome inhibitor) on c-Myc expression. The effects of 20 μmol/L juglone on the proliferation and apoptosis of HeLa cells with RNA interference-mediated knockdown of c-Myc were evaluated with MTT assay and flow cytometry.@*RESULTS@#Treatment with juglone significantly lowered c-Myc protein expression in HeLa cells in a concentration-and time-dependent manner (P < 0.05). Juglone obviously shortened the half-life of c-Myc protein, and the addition of MG132 significantly up-regulated the expression level of c-Myc protein (P < 0.05). Juglone treatment also promoted ubiquitination of c-Myc protein in HeLa cells. Compared with the cells transfected with a negative control construct, the cells transfected with si-c-Myc showed significantly decreased proliferation inhibition and a lowered cell rate with early apoptosis after juglone treatment (P < 0.05).@*CONCLUSION@#Juglone inhibits proliferation and promotes apoptosis of HeLa cells by affecting the ubiquitination of c-Myc protein.

Construction of MicroRNA-Target Interaction Networks Based on MicroRNA Expression Profiles of HRV16-infected H1-HeLa Cells / 生物医学与环境科学（英文）

In the present study we investigated the changes in miRNA levels inhuman rhinovirus 16 (HRV16)-infected cells. A small RNA deep sequencing experiment was performed through next-generation sequencing. In total, 53 differentially expressed miRNAs were confirmed by RT-qPCR, including 37 known miRNAs and 16 novel miRNAs. Interaction networks between differentially expressed miRNAs and their targets were established by mirDIP and Navigator. The prediction results showed that QKI, NFAT5, BNC2, CELF2, LCOR, MBNL2, MTMR3, NFIB, PPARGC1A, RSBN1, TRPS1, WDR26, and ZNF148, which are associated with cellular differentiation and transcriptional regulation, were recognized by 12, 11, or 9 miRNAs. Many correlations were observed between transcriptional or post-transcriptional regulation of an miRNA and the expression levels of its target genes in HRV16-infected H1-HeLa cells.

SET8 Inhibition Potentiates Radiotherapy by Suppressing DNA Damage Repair in Carcinomas / 生物医学与环境科学（英文）

Objective@#SET8 is a member of the SET domain-containing family and the only known lysine methyltransferase (KMT) that monomethylates lysine 20 of histone H4 (H4K20me1). SET8 has been implicated in many essential cellular processes, including cell cycle regulation, DNA replication, DNA damage response, and carcinogenesis. There is no conclusive evidence, however, regarding the effect of SET8 on radiotherapy. In the current study we determined the efficacy of SET8 inhibition on radiotherapy of tumors and the underlying mechanism.@*Methods@#First, we explored the radiotherapy benefit of the SET8 expression signature by analyzing clinical data. Then, we measured a series of biological endpoints, including the xenograft tumor growth in mice and apoptosis, frequency of micronuclei, and foci of 53BP1 and γ-H2AX in cells to detect the SET8 effects on radiosensitivity. RNA sequencing and subsequent experiments were exploited to verify the mechanism underlying the SET8 effects on radiotherapy.@*Results@#Low expression of SET8 predicted a better benefit to radiotherapy in lung adenocarcinoma (LUAD) and invasive breast carcinoma (BRCA) patients. Furthermore, genetic deletion of SET8 significantly enhanced radiation treatment efficacy in a murine tumor model, and A549 and MCF7 cells; SET8 overexpression decreased the radiosensitivity. SET8 inhibition induced more apoptosis, the frequency of micronuclei, and blocked the kinetics process of DNA damage repair as 53BP1 and γ-H2AX foci remained in cells. Moreover, RNF8 was positively correlated with the SET8 impact on DNA damage repair.@*Conclusion@#Our results demonstrated that SET8 inhibition enhanced radiosensitivity by suppressing DNA damage repair, thus suggesting that SET8 potentiated radiotherapy of carcinomas. As new inhibitors of SET8 are synthesized and tested in preclinical and clinical settings, combining SET8 inhibitors with radiation warrants consideration for precise radiotherapy.

The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

Analysis of PKD2 gene variant and protein localization in a pedigree affected with polycystic kidney disease / 中华医学遗传学杂志

OBJECTIVE@#To detect the mutation site in a pedigree affected with autosomal dominant polycystic kidney disease (ADPKD) and verify its impact on the protein function.@*METHODS@#Peripheral blood samples were collected from the proband and his pedigree members for the extraction of genomic DNA. Mutational analysis was performed on the proband through whole-exome sequencing. Suspected variant was verified by Sanger sequencing. A series of molecular methods including PCR amplification, restriction enzyme digestion, ligation and transformation were also used to construct wild-type and mutant eukaryotic expression vectors of the PKD2 gene, which were transfected into HEK293T and HeLa cells for the observation of protein expression and cell localization.@*RESULTS@#The proband was found to harbor a c.2051dupA (p. Tyr684Ter) frame shift mutation of the PKD2 gene, which caused repeat of the 2051st nucleotide of its cDNA sequence and a truncated protein. Immunofluorescence experiment showed that the localization of the mutant protein within the cell was altered compared with the wild-type, which may be due to deletion of the C-terminus of the PKD2 gene.@*CONCLUSION@#The c.2051dupA (p. Tyr684Ter) mutation of the PKD2 gene probably underlay the pathogenesis of ADPKD in this pedigree.

Preparation of paclitaxel-loaded and folic acid-modified poly (lactic-co-glycolic acid) nano-micelles and in vitro anticancer effect on cervical cancer HeLa cells / 中国中药杂志

The paclitaxel-loaded and folic acid-modified poly(lactic-co-glycolic acid) nano-micelles(PTX@FA-PLGA-NMs) were prepared by the emulsion solvent evaporation method, and the parameters of paclitaxel-loaded nano-micelles were optimized with the particle size and PDI as evaluation indexes. The morphology of the nano-micelles was observed by transmission electron microscopy(TEM), and the stability, drug loading and encapsulation efficiency were systematically investigated. In vitro experiments were performed to study the cytotoxic effects of nano-micelles, apoptosis, and cellular uptake. Under the optimal parameters, the nano-micelles showed the particle size of(125.3±1.2) nm, the PDI of 0.086±0.026, the zeta potential of(-20.0±3.8) mV, the drug loading of 7.2%±0.75%, and the encapsulation efficiency of 50.7%±1.0%. The nano-micelles were in regular spherical shape as observed by TEM. The blank FA-PLGA-NMs exhibited almost no inhibitory effect on the proliferation and growth of tumor cells, while the drug-loaded nano-micelles and free PTX exhibited significant inhibitory effects. The IC_(50) of PTX@FA-PLGA-NMs and PTX was 0.56 μg·mL~(-1) and 0.66 μg·mL~(-1), respectively. The paclitaxel-loaded nano-micelles were potent in inhibiting cell migration as assessed by the scratch assay. PTX@FA-PLGA-NMs had good pro-apoptotic effect on cervical cancer HeLa cells and significantly promoted the uptake of HeLa cells. The results of in vitro experiments suggested that PTX@FA-PLGA-NMs could target and treat cervical cancer HeLa cells. Therefore, as nanodrug carriers, PTX@FA-PLGA-NMs with anti-cancer activity are a promising nano-system for improving the-rapeutic effects on tumors.

LINC00665 promotes HeLa cell proliferation, migration, invasion and epithelial-mesenchymal transition by activating the WNT-CTNNB1/β‑catenin signaling pathway / 生理学报

There is increasing evidence that long non-coding RNA (lncRNA) plays critical roles in cancer progression. However, the role of long non-coding RNA 00665 (LINC00665) in most cancers is poorly understood. The purpose of the present study was to reveal the functional role of LINC00665 in cervical cancer cells. HeLa cells were subjected to LINC00665 short hairpin RNA (shRNA) or control shRNA treatment to investigate the metastasis and proliferation phenotype of cervical cancer cells in vitro and in vivo. Transcriptome sequencing experiments of HeLa cells in LINC00665 silencing or control group were conducted, and the differentially expressed genes (DEGs) were screened. The DEGs were subjected to Metascape database functional analysis and gene set enrichment analysis. Epithelial-mesenchymal transition (EMT) related markers and a key element of WNT/β‑catenin pathway, CTNNB1 (catenin beta 1), were detected by Western blot and immunofluorescence assay. The results showed that silencing LINC00665 reduced cell viability of Hela cells, up-regulated protein expression level of E-cadherin, down-regulated protein expression levels of N-cadherin, Vimentin and CTNNB1, and inhibited cell migration and invasion of HeLa cells. Bioinformatics analysis results showed that LINC00665 might promote EMT by activating WNT-CTNNB1/β‑catenin signaling pathway. These results indicate that LINC00665 has functions in transcriptional EMT regulation via WNT-CTNNB1/β‑catenin signaling pathway and therefore can be developed as a therapeutic target for cervical cancer.

The anti-neoplastic activities of aloperine in HeLa cervical cancer cells are associated with inhibition of the IL-6-JAK1-STAT3 feedback loop / 中国天然药物

Cervical cancer (CC) is recognized as the most common neoplasm in the female reproductive system worldwide. The lack of chemotherapeutic agents with outstanding effectiveness and safety severely compromises the anti-cipated prognosis of patients. Aloperine (ALO) is a natural quinolizidine alkaloid with marked anti-cancer effects on multiple malignancies as well as favorable activity in relieving inflammation, allergies and infection. However, its therapeutic efficacy and underlying mechanism in CC are still unclear. In the current study, MTT assay was employed to evaluate the viability of HeLa cells exposed to ALO to preliminarily estimate the effectiveness of ALO in CC. Then, the effects of ALO on the proliferation and apoptosis of HeLa cells were further investigated by plate colony formation and flow cytometry, respectively, while the migration and invasion of ALO-treated HeLa cells were evaluated using Transwell assay. Moreover, nude mice were subcutaneously inoculated with HeLa cells to demonstrate the anti-CC properties of ALO in vivo. The molecular mechanisms underlying these effects of ALO were evaluated by Western blot and immunohistochemical analysis. This study experimentally demonstrated that ALO inhibited the proliferation of HeLa cells via G2 phase cell cycle arrest. Simultaneously, ALO promoted an increase in the percentage of apoptotic HeLa cells by increasing the Bax/Bcl-2 ratio. Additionally, the migration and invasion of HeLa cells were attenuated by ALO treatment, which was considered to result from inhibition of epithelial-to-mesenchymal transition. For molecular mechanisms, the expression and activation of the IL-6-JAK1-STAT3 feedback loop were markedly suppressed by ALO treatment. This study indicated that ALO markedly suppresses the proliferation, migration and invasion and enhances the apoptosis of HeLa cells. In addition, these prominent anti-CC properties of ALO are associated with repression of the IL-6-JAK1-STAT3 feedback loop.

Altered Splicing in Stable Cell Strains Expressing Mini-hF9 Gene with Nonsense Mutation / 中国实验血液学杂志

OBJECTIVE@#To investigate the molecular mechanism in stable cell strains expressing Mini-hF9 gene with nonsense mutation.@*METHODS@#Mini-hF9 gene and its nonsense mutants were transfected into HeLa cells independently, and stable cell strains were obtained after G418 resistance screening and monoclonal transformation. The altered splicing and protein expression of mRNA in Mini-hF9 gene in stable cell strains were detected by using RT-PCR and Western blot.@*RESULTS@#The wild type and nonsense mutated human coagulation factor IX stable cell strains were constructed successfully, which were named HeLa-F9-WT, HeLa-F9-M1 and HeLa-F9-M2. Only normal splicing Norm was detected in the wild-type cell strain HeLa-F9-WT; Norm and Alt-S1 splicing were detected in HeLa-F9-M1; while Norm, Alt-S1 and Alt-S2 splicing were detected in HeLa-F9-M2.@*CONCLUSION@#The nonsense associated altered splicing (NAS) pathway, which generated alternately spliced transcripts, might be triggered in coagulation factor IX gene with nonsense mutation.

7SK truncation at 128-179 nt suppresses embryonic stem cell proliferation / 南方医科大学学报

OBJECTIVE@#To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell (ESCs) proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism (PD).@*METHODS@#ESC line R1 was transfected with the CRISPR/Cas9 system, and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations. A lentivirus system was used to knock down cyclin-dependent kinase 9 (CDK9) in clones with 7SK deletion mutations, and the effect of CDK9 knockdown on the protein level of cell division cycle 6 (CDC6) was analyzed with Western blotting.@*RESULTS@#We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs, which resulted in deficiency of cell proliferation. 7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7 (LARP7) and CDC6.@*CONCLUSIONS@#7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6. 7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity, suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.

Isolation of Polyphenols from Soursop Annona muricata L Leaves Using Green Chemistry Techniques and their Anticancer Effect

HIGHLIGHTS Isolate, fractionate and characterize extracts obtained from soursop leaves. Use of emerging green technologies such as microwave-ultrasound hybridization. The extracts contain kaempferol, procyanidins, catechin, and quercetin. The total ethanolic extract demonstrates cytotoxic effect on HeLa cells.

Abstract Cervical cancer is classified as the fourth most common malignancy in women. Natural compounds are a therapeutic alternative in cancer therapy. The aim of the study is to isolate, fractionate, and characterize extracts obtained from soursop leaves (Annona muricata L.) and determine their cytotoxic effect against HeLa cervical cancer cells and non-carcinogenic fibroblast 3T3 cells. The phytochemicals of soursop leaves were extracted through emerging green technologies such as the novel use of microwave-ultrasound hybridization and the use of environmentally friendly solvents (water and ethanol), in addition to the purification of extracts enriched in polyphenols by liquid chromatography with Amberlite XAD-16. Total aqueous and ethanolic extract were purified, as well as the fraction one of each extract. The extracts recovered from soursop leaves contained kaempferol and its isomers, procyanidins, catechin, and quercetin. The viability of the cells was determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. HeLa and 3T3 cells were exposed to concentrations of 25, 50, 75, 100, 150, 200, and 250 ppm of a solution of soursop leaf extract powder. The MTT assay showed that soursop leaf extracts were toxic to both cell lines in general, however, the ethanolic extract at 25 and 50 ppm demonstrated inhibition in cell viability against the HeLa cancer line and low cytotoxicity for 3T3 fibroblast cells. In conclusion, the novel microwave-ultrasound hybridization technology allows the extraction of polyphenols that may have a potential cytotoxic effect on cancer cells.

A new cytotoxic isoflavane from Dalbergiae Odoriferae Lignum / 中国中药杂志

Fourteen compounds were isolated from the ethanol extract of Dalbergiae Odoriferae Lignum by various chromatographic techniques, including column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. Their structures were determined by spectroscopic techniques as S-3'-hydroxy-7,2',4'-trimethoxyisoxane(1), 2-(2',4'-dimethoxyphenyl)-6-hydroxybenzofuran(2), 2-(2'-hydroxy-4'-methoxyphenyl)-6-methoxybenzofuran(3), 7,2',4'-trimethoxydihydroisoflavone(4), sativanone(5), 3,9-dimethoxy-6H-benzofuro[3,2-c]chromen-6-one(6),(6 aS,11 aS)-homopterocarpin(7),(6 aS,11 aS)-8-hydroxy-3,9-dimethoxypterocarpan(8),(6 aS,11 aS)-3,8,9-trimethoxypterocarpan(9), isodalbergin(10), isoliquiritigenin(11), butein(12), butin(13) and 3,7,4'-trihydroxyflavone(14). Among them, compound 1 was a new compound, while 2 and 3 were new natural products, 6, 8, 9 and 14 were isolated for the first time from Dalbergiae Odoriferae Lignum. Compounds 1-14 were tested for their cytotoxic activity against human hepatoma cell line BEL-7402, human gastric cancer cell line SCG-7901, human lung cancer cell line A549, human chronic myeloid leukemia cell line K562 and HeLa human cervical cancer cellline by MTT method. Compound 1 exhibited significant cytotoxicity with IC_(50) values ranging from 2.85 to 11.62 μg·mL~(-1). In addition, 2, 11 and 12 showed weak cytotoxic activities.

SULF2 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells through the ERK/AKT signaling pathway

The objective of this study was to explore the role of the SULF2-mediated ERK/AKT signaling pathway in cervical cancer. SULF2 expression was detected in tumor tissues and tumor-adjacent normal tissues from cervical cancer patients. HeLa cells were divided into six groups: control group, NC group, SULF2 siRNA group, SULF2 group, SULF2 + LY294002 group, and SULF2 + U0125 group. In each group, HeLa cells received the corresponding treatment, followed by measurement of the cellular biological characteristics and expression of the ERK/AKT signaling pathway. We also confirmed the effect of SULF2 in vivo using a xenograft model in nude mice. SULF2 was upregulated in cervical cancer tissues, which was specifically associated with the clinical stage, histological differentiation, and lymphatic metastasis. Compared to the control group, the SULF2 siRNA group displayed decreased expression of SULF2, concomitant with reduced proliferation, migration, and invasion, but there was an increase in the apoptosis rate of HeLa cells, as well as downregulation of the p-Akt/Akt, p-ERK/ERK, and Bax/Bcl-2 ratios and cyclin D1. Additionally, tumor growth was significantly inhibited in the xenograft model of nude mice. The results in the SULF2 group were quite the opposite in which SULF2 facilitated the growth of cervical cancer cells, which was reversed by LY294002 or U0126. SULF2 is highly expressed in cervical cancer, and thus, downregulation of SULF2 can inhibit the ERK1/2 and AKT signaling pathways to suppress the proliferation, invasion, and migration of cervical cancer cells while facilitating apoptosis.

The extraordinary trajectory of the Papillomavirus through human history / A extraordinária trajetória do Papilomavirus através da história da humanidade

Papillomaviruses have been infecting people since the beginning of human life on earth. The most relevant chapters of this story were written by the brilliant, diverse, and prominent scientists of their respective times. However, an important part of this story is the Papillomavirus victims and their adverse situations. There have also been disputes over intellectual primacy of the discoveries and the collaborators who were not given the recognition according to the role they played. This article will guide the reader through the remarkable facts of this conflicting and interesting relationship between humans and the Papillomavirus

O papilomavírus tem infectado pessoas desde o começo da vida humana na Terra. Os capítulos mais relevantes dessa história foram escritos por mãos brilhantes de diversos e proeminentes cientistas em seus respectivos tempos. No entanto, parte importante dessa história também foi construída por vítimas do papilomavírus e suas situações adversas. Houve também disputas sobre a primazia intelectual das descobertas e os colaboradores cujo reconhecimento é menor que o papel que desempenhavam. Este artigo levará o leitor por um breve passeio pelos fatos marcantes dessa conflituosa e interessante relação entre o homem e o papilomavírus

Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

Investigating the Sonodynamic-Radiosensitivity Effect of Gold Nanoparticles on HeLa Cervical Cancer Cells

BACKGROUND: In this article, we estimated the combined effect of radiotherapy (RT) with ultrasound (US) wave and the ability of gold nanoparticles (GNPs) to improve their combined therapeutic effects. METHODS: At first, HeLa cells received the various treatment modalities: RT (6 MV; 0.5, 1, and 2 Gy), US irradiation (1 MHz; 0.5, 1, and 1.5 W/cm2, 1 minute), and RT+US. Afterwards, the enhanced effect of US on RT was evaluated. Then, the effect of the synthesized GNPs at different concentrations (0.2, 1, and 5 µg/mL, 24 hours) was evaluated to assess the effect on HeLa cells combined with RT+US. Cell survival rates in the different treatment groups at 24, 48, and 72 hours post-treatment were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays. RESULTS: Our results show US irradiation could enhance the effect of RT at the same radiation dose and could be utilized as a sensitizer agent for RT. Moreover, our findings indicate RT+US in combination with different nanoparticle concentrations could enhance the effect of RT+US so that they can improve the treatment results up to 9.93 times and act as sonodynamic-radiosensitivity. These results also indicate that the combination of RT with US along with GNPs has synergistic effects compared to RT or US alone. Cell survival results show that combining the low US waves (1.5 W/cm2), GNPs (5 μg/mL), and X-rays (2 Gy) increase the cytotoxicity on HeLa cell up to 95.8%. CONCLUSION: We concluded that GNPs could act as a good sensitizing agent in RT+US irradiation and could result in the synergistic effects.

Pseudolaric acid B inhibits PAX2 expression through Wnt signaling and induces BAX expression, therefore promoting apoptosis in HeLa cervical cancer cells / 부인종양

OBJECTIVES: Pseudolaric acid B (PAB) has been shown to inhibit the growth of various tumor cells, but the molecular details of its function are still unknown. This study investigated the molecular mechanisms by which PAB induces apoptosis in HeLa cells. METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed to investigate the effect of PAB treatment in various cervical cancer cell lines. Annexin V/propidium iodide staining combined with flow cytometry and Hoechst 33258 staining were used to assess PAB-induced apoptosis. Additionally, we performed bioinformatics analyses and identified a paired box 2 (PAX2) binding site on the BAX promoter. We then validated the binding using luciferase and chromatin immunoprecipitation assays. Finally, western blotting assays were used to investigate PAB effect on the Wnt signaling and the involved signaling molecules. RESULTS: PAB promotes apoptosis and downregulates PAX2 expression in HeLa cells in a time- and concentration-dependent manner. PAX2 binds to the promoter of BAX and inhibits its expression; therefore, PAX2 inhibition is associated with increased levels of BAX, which induces apoptosis of HeLa cells via the mitochondrial pathway. Additionally, PAB inhibits classical Wnt signaling. CONCLUSION: PAB effectively inhibits Wnt signaling and PAX2 expression, and increases BAX levels, which induce apoptosis in HeLa cells. Therefore, PAB is a promising natural molecule for the treatment of cervical cancer.